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CELLULAR AND MOLECULAR

Dissimilar Pharmacological Responses by a New Series of Imidazoline Derivatives at Precoupled and Ligand-Activated _2A-Adrenoceptor States: Evidence for Effector Pathway-Dependent Differential Antagonism

Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, Castres, France

Received December 16, 2002; accepted March 7, 2003.

	Abstract

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Whereas agonist-directed differential signaling at a single receptor subtype has become an accepted pharmacological concept, distinct behaviors by ligands that are assumed to be antagonists is less documented. The intrinsic activity and capacity of antagonism for a new series of imidazoline-derived adrenergic ligands analogous to dexefaroxan were investigated by measuring two distinct signaling pathways at the recombinant human _2A-adrenoceptor (_2A AR): 1) pertussis toxin-resistant guanosine 5'-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPS) binding responses mediated by either a recombinant G_oCys³⁵¹Ile or G_i2Cys³⁵²Ile protein in CHO-K1 cells, and 2) inhibition of cAMP formation in a stably transfected C6-glial cell line. Ligands could be differentiated as inverse agonists [i.e., 2-(4-methoxy-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 851062], neutral antagonists [i.e., 2-(4-hydroxy-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 851057], partial [i.e., 2-(4-chloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 821008], and high-efficacy [i.e., 2-(6,7-dichloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 821010] agonists at a precoupled _2A AR state in the copresence of a G_oCys³⁵¹Ile protein but not G_i2Cys³⁵²Ile protein by monitoring [³⁵S]GTPS binding responses. Neither positive nor negative efficacy was observed for these compounds by monitoring the adenylate cyclase pathway at a presumably low-affinity _2A AR state. The capacity of the dexefaroxan analogs to antagonize the (-)-epinephrine-mediated [³⁵S]GTPS binding response at a G_oCys³⁵¹Ile protein was inversely correlated with their magnitude of intrinsic activity and unrelated to their ligand binding affinity for the _2A AR. On the other hand, their capacity to antagonize either (-)-epinephrine or 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline tartrate (UK 14304)-mediated inhibition of forskolin-stimulated cAMP formation was not related with the rank order of antagonist capacity for the (-)-epinephrine-mediated [³⁵S]GTPS binding response. In conclusion, these data demonstrate that certain ₂ AR ligands that are assumed to be antagonists, may yield dissimilar pharmacological responses, dependent on the investigated agonist-stimulated effector pathway.

The concept that ₂ ARs assume different conformations that can selectively and differentially couple them to specific second messenger pathways received support from mutagenesis studies (Wang et al., 1991; Lakhlani et al., 1996; Rudling et al., 1999). Evidence for agonist-specific _2A AR-mediated responses has also been obtained for the wild-type (wt) _2A AR using different natural and synthetic agonists, such as catecholamines, imidazolines, and azepines (Eason et al., 1994; Airriess et al., 1997). These data suggest multiple mechanisms of _2A AR activation by different classes of agonists. It is reasonable to assume that agonist-specific coupling of the _2A AR to intracellular pathways may underline differential signaling. Most likely, distinct conformations of the activated agonist receptor complex (Kenakin, 1995) may be responsible for the observed specific responses. Marjamäki et al. (1999) have combined targeted mutagenesis experiments with structural modeling to show that two agonists that covalently bind to _2A ARs, chloroethylclonidine and 2-aminoethyl methanethiosulfate, recognize two different receptor conformations. Recently, differential signaling on G_i/o protein-mediated _2A AR responses in HEL 92.1.7 cells has been demonstrated (Kukkonen et al., 2001). Marked differences in the potencies of agonists to mediate elevation of Ca²⁺ mobilization and inhibition of forskolin-induced cAMP stimulation were observed. These results further suggest that ligand-dependent _2A AR states produced by catecholamines compared with other classes of agonists are different and, therefore, may be able to preferentially activate different signaling pathways. The observed spectrum of intrinsic activities for a series of ligands at the _2A AR suggests that most commonly investigated antagonists behave as either inverse agonists or partial agonists (Pauwels et al., 2000; Wade et al., 2001). This wide spectrum of intrinsic activities becomes more apparent when measuring activity at facilitating mutant _2A ARs (Pauwels and Colpaert, 2000).

The imidazoline derivative dexefaroxan behaves as a selective ₂ AR antagonist in in vivo studies (Martel et al., 1998; Tellez et al., 1999), but its in vitro intrinsic activity seems more dependent on the experimental assay system. Dexefaroxan acted as a silent antagonist when monitoring Ca²⁺ responses mediated by wt _2A, _2B, and _2C ARs in the copresence of a G₁₅ protein (Pauwels et al., 2001). Under conditions where enhanced constitutive activation was observed by increasing the proportion of high-affinity _2A AR* states (i.e., mutant Thr³⁷³Lys _2A AR in the copresence of a G_oCys³⁵¹Ile protein; Pauwels et al., 2000), dexefaroxan showed a nonsignificant tendency to decrease the basal [³⁵S]GTPS binding response. In contrast, several facilitating mutations, such as Asp⁷⁹Asn, Ser²⁰⁰Ala, or Ser²⁰⁴Ala in the _2A AR generated receptor states at which dexefaroxan yielded positive efficacy when the mutant _2A ARs were fused to a G₁₅ protein (i.e., +45% versus (-)-epinephrine at the mutant Ser²⁰⁴Ala _2A AR; Pauwels and Colpaert, 2000). In the present study, we further investigated the intrinsic activity for a new series of ₂ AR ligands based on the imidazoline derivative dexefaroxan, under experimental conditions of either low or high constitutive _2A AR activation, by favoring respectively low- and high-affinity receptor states. The antagonist capacity of these compounds was also determined in CHO-K1 cells by measuring a pertussis toxin (PTX)-resistant, (-)-epinephrine-stimulated [³⁵S]GTPS binding response (Dupuis et al., 1999) under experimental conditions favoring or not high-affinity _2A AR* states, as well as a PTX-sensitive, agonist-mediated inhibition of forskolin-induced cAMP formation in a C6-glial cell line stably expressing _2A ARs. The antagonist data with the dexefaroxan analogs for the [³⁵S]GTPS binding and cAMP responses demonstrate dissimilar pharmacological profiles at an agonist-activated _2A AR state.

	Materials and Methods

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Construction of _2A AR and G Protein Genes. The human wt _2A AR (R.C. 2.1.ADR.A2A, GenBank accession no. M23533 [GenBank] ), rat G_oCys³⁵¹Ile protein (based on GenBank accession no. M17526 [GenBank] ), and rat G_i2Cys³⁵²Ile (based on GenBank accession no. M17528 [GenBank] ) genes were cloned by polymerase chain reaction as described previously (Wurch et al., 1999) and separately ligated into a pCR3.1 mammalian expression vector. Cloned cDNAs were fully sequenced on an ABI Prism 310 genetic analyzer by using a dichlororhodamine cycle sequencing kit confirming the respective nucleotide sequences.

Synthesis of Dexefaroxan Analogs. Synthesis and purification of the dexefaroxan analogs were performed according to the procedures initially described by Chapleo et al. (1983, 1984).

Receptor Binding Assay to _2A-Adrenoceptors. Membrane preparations of CHO-K1 cells transiently cotransfected with _2A AR and G_oCys³⁵¹Ile protein plasmids were prepared in 50 mM Tris-HCl, pH 7.6, as described previously (Wurch et al., 1999). Incubation mixtures consisted of 0.4 ml of cell membranes (30 µg of protein), 0.05 ml of radioligand [(1,4-[6,7(n)-[³H]benzodioxan-2-methoxy-2-yl)-2-imidazoline hydrochloride (RX 821002), 1 nM] and 0.05 ml of compound or phentolamine (10 µM) to determine nonspecific binding. The reactions were stopped after a 30-min incubation at 25°C by adding 3.0 ml of ice-cold 50 mM Tris-HCl, pH 7.6, and rapid filtration over GF/B glass fiber filters (Whatman, Maidstone, UK) using a harvester (Brandel Inc., Gaithersburg, MD), washed, and counted as described in Wurch et al. (1999). Scatchard analysis for these transfected cells yielded the following binding parameters using [³H]RX 821002 as a radioligand: K_d of 1.19 ± 0.12 nM and B_max of 4.16 ± 0.30 pmol/mg of protein. IC₅₀ values for ligands as obtained from competition binding curves performed over a range of six concentrations were converted into pK_i values as described previously (Wurch et al., 1999).

cAMP Measurements. C6-glial cells (CCL-107; American Type Culture Collection, Manassas, VA) stably expressing a wt _2A AR ([³H]RX 821002; K_d of 0.96 ± 0.09 nM and B_max of 638.5 ± 89.5 fmol/mg protein) were prepared as a monoclonal cell line, cultured as described previously (Pauwels et al., 1996), and used for cAMP measurements. Cultures were incubated for 15 min at 37°C with 1.0 ml of controlled salt solution containing 1 mM isobutylmethylxanthine in the presence of 100 µM forskolin either in the absence or presence of 10 nM (-)-epinephrine ± 1 µM propranolol or 10 nM UK 14304 to determine maximal cAMP inhibition. Antagonist (1 µM) was coincubated with 10 nM of agonist to determine its antagonist capacity. The reaction was stopped by the addition of 0.1 ml of HClO₄ to a final concentration of 0.04 M and afterwards neutralized. The cellular cAMP content was assayed by a radioimmunoassay kit. Antagonism of either UK 14304 or (-)-epinephrine-mediated inhibition of forskolin-induced cAMP formation was calculated as a percentage of the inhibition obtained with 1 µM (+)-2-(2-ethoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole (RX 811059). Control experiments performed with nontransfected C6-glial cells did not show UK14304 (10 nM)-mediated inhibition of 100 µM forskolin-stimulated cAMP formation.

[³⁵S]GTPS Binding Responses. CHO-K1 cells grown to 60 to 80% confluence were used for transfection using a LipofectAMINE Plus kit. Three micrograms of a pCR3.1 plasmid containing a wt _2A AR cDNA without or with either a G_oCys³⁵¹Ile or a G_i2Cys³⁵²Ile cDNA plasmid (3 µg) was mixed with 10 µl of LipofectAMINE Plus reagent in 0.2 ml of Opti-MEM and incubated at room temperature for 15 min. Subsequently, 20 µl of LipofectAMINE reagent diluted in 0.2 ml of Opti-MEM was added for 15 min and exposed with 5 ml of Opti-MEM to CHO-K1 cells for 3 h at 37°C. Thereafter, cells were further incubated with 10 ml of complete growth medium and harvested 48 h after transfection. Treatment with PTX (20 ng/ml) was performed overnight before membranes were prepared. Agonist-independent (basal) and agonist-dependent [³⁵S]GTPS binding responses (Pauwels et al., 2000) were performed to a membrane preparation in 20 mM HEPES, pH 7.4, supplemented with 30 µM GDP, 100 mM KCl, 3 mM MgCl₂, and 2 mM ascorbic acid. [³⁵S]GTPS binding responses were systematically performed in parallel with 1 µM RX 811059 to define the magnitude of inverse agonism. Maximal stimulation of [³⁵S]GTPS binding was defined in the presence of 10 µM (-)-epinephrine and calculated versus basal [³⁵S]GTPS binding, unless otherwise indicated. pEC₅₀ and pIC₅₀ values represent the ligand concentrations that showed, respectively, 50% stimulation and inhibition of its own maximal modulation of basal [³⁵S]GTPS binding. In antagonist experiments, putative antagonists (1 µM) were coincubated with (-)-epinephrine (10 nM).

Protein Content. Membrane protein levels were estimated with a dye-binding assay using a Bio-Rad (Hercules, CA) kit; bovine serum albumin was used as a standard (Bradford, 1976).

Statistical Analysis. Statistics on E_max values for [³⁵S]GTPS binding and cAMP responses were determined using a Student's t test for two group comparisons. Correlations were performed using a Pearson's correlation test.

Materials. The pCR3.1 vector, the LipofectAMINE Plus kit, cell culture media, fetal calf serum, culture plates, and Bordetella pertussis toxin (PTX, 50 µg/ml) were obtained from Invitrogen (San Diego, CA). The ABI Prism 310 genetic analyzer and the dichlororhodamine terminator cycle sequencing kit were purchased from Applied Biosystems (Foster City, CA). C6-glial and CHO-K1 cells were obtained from American Type Culture Collection. [³H]RX 821002 (50 Ci/mmol) and [³⁵S]GTPS (1035–1163 Ci/mmol) were obtained from Amersham Biosciences Inc. (Les Ulis, France). The cAMP radioimmunoassay kit was from Immunotech (Marseille, France). (-)-Epinephrine was from Sigma-Aldrich (St. Louis, MO). The investigated ₂ AR ligands (Table 1) were prepared intramuros. Stock solutions of ligands were prepared at 10^-3 M. Serial dilutions were made in the respective incubation buffer.

View this table: [in this window] [in a new window]   TABLE 1 Chemical structures, binding affinities, and intrinsic activities of 2 ligands for 2A AR-mediated [35S]GTPS binding and inhibition of forskolin-stimulated cAMP responses

	Results

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Binding Affinities of Dexefaroxan Analogs at _2A ARs. Binding affinities for a series of dexefaroxan analogs as obtained by inhibition of [³H]RX 821002 binding were determined at CHO-K1 cellular membranes containing recombinant wt _2A AR and G_oCys³⁵¹Ile proteins (Table 1). The investigated series of ligands covered a large window of binding affinities from the nanomolar (i.e., RX 811059 and RX 821008) to the micromolar range [i.e., UK 14304, (-)-epinephrine]. Replacement of the 2-ethyl group of dexefaroxan by either a 2-propyl (RX 831001) or 2-n-pentyl group (RX 831003) did almost not affect their binding affinity compared with dexefaroxan. The coaddition of either a 4-hydroxy (RX 851057), 4-methoxy (RX 851062), or a 5-chloro substituent (RX 841047) attenuated about 10-fold their binding affinity. A chloro-substitution at position 4 of the dihydrobenzofuran (RX 821008) instead of an ethyl-substitution at position 2 of dexefaroxan displayed a 2.5-fold higher binding affinity. Addition of a chloro-molecule at position 5 (RX 811012) or 5 and 7 (RX 821022) of the dihydrobenzofuran moiety of dexefaroxan attenuated the binding affinity by a factor 8 and 24, respectively. Atipamezole, an imidazole derivative distantly related to dexefaroxan, displayed a similar binding affinity as dexefaroxan. Similar binding affinities were obtained at membranes of C6-glial cells stably expressing a wt _2A AR (data not shown).

Intrinsic Activity of Dexefaroxan Analogs at the wt _2A AR. Constitutive _2A AR activation was observed in CHO-K1 cells by coexpression with PTX-resistant G_oCys³⁵¹Ile and G_i1Cys³⁵¹Ile proteins in contrast to the mutant G_i2Cys³⁵²Ile and G_i3Cys³⁵¹Ile proteins (Pauwels et al., 2000). Recombinant G_oCys³⁵¹Ile and G_i2Cys³⁵²Ile proteins were further investigated because both yielded sufficient stimulation by (-)-epinephrine (10 µM) to evaluate antagonist capacities (respectively, 84.2 ± 9.3 and 52.5 ± 0.5% versus basal [³⁵S]GTPS binding responses). Figure 1 shows the spectrum of intrinsic activities for dexefaroxan and its analogs, (+)-RX 811059 and atipamezole at 1 µM for the wt _2A AR in the copresence of either a G_oCys³⁵¹Ile or G_i2 Cys³⁵²Ile protein as measured at the level of [³⁵S]GTPS binding. The ligands could be differentiated in three subgroups on the basis of either negative efficacy, silent activity, or positive efficacy in the presence of a G_oCys³⁵¹Ile protein. The potencies of RX 821010 (efficacious agonist; pEC₅₀ = 7.19 ± 0.12) and RX 851062 (inverse agonist; pIC₅₀ = 7.25 ± 0.5) fitted with their respective binding affinity (Table 1) and could be fully blocked by the silent, neutral antagonist atipamezole (1 µM; Fig. 2). In contrast, none of the investigated compounds yielded inverse agonist properties in the copresence of a G_i2Cys³⁵²Ile protein; the dexefaroxan analogs behaved either as silent compounds or weak partial agonists (Fig. 1). RX 821010, which yielded high-efficacy agonist properties in the copresence of a G_oCys³⁵¹Ile protein, reached only 44% of the maximal [³⁵S]GTPS binding response induced by (-)-epinephrine at a G_i2Cys³⁵²Ile protein (Fig. 1). Otherwise, this series of investigated compounds tested in the absence of agonist were, with exception of dexefaroxan (78.9 ± 3.1% versus 100 µM forskolin), unable to modulate significantly forskolin (100 µM)-stimulated cAMP production in C6-glial cells stably expressing the _2A AR (Table 1).

View larger version (24K): [in this window] [in a new window]   Fig. 1. Emax values of 2A AR ligands' [35S]GTPS binding response mediated by wt 2A AR in the copresence of either a GoCys351Ile or Gi2Cys352Ile protein in CHO-K1 cells. CHO-K1 cells were transfected and treated with PTX (20 ng/ml), as described under Materials and Methods. [35S]GTPS binding responses were performed with 0.5 nM [35S]GTPS and 1 µM of each ligand except (-)-epinephrine (10 µM). Emax values are expressed in percentage versus basal [35S]GTPS binding response (152 ± 16 and 142 ± 13 fmol/mg protein for, respectively, GoCys351Ile and Gi2Cys352Ile proteins). Bar graphs correspond to mean values ± S.E.M. of a minimum of nine (GoCys351Ile protein, ) and three (Gi2Cys352Ile protein, ) independent experiments.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Antagonism of RX 821010- and RX 851062-mediated [35S]GTPS binding responses by atipamezole at the wt 2A AR coexpressed with a GoCys351Ile protein in CHO-K1 cells. Cultures were treated overnight with PTX (20 ng/ml) and assayed for [35S]GTPS binding responses, as indicated under Materials and Methods. Concentration binding curves are constructed using mean values ± S.E.M. from six independent transfection experiments, each one performed in duplicate. A, antagonism of RX 821010-mediated [35S]GTPS binding response by atipamezole. pEC50 RX 821010, 7.19 ± 0.12; pEC50 RX 821010 + 1 µM atipamezole, <5.00. B, antagonism of RX 851062-mediated [35S]GTPS binding response by atipamezole. pIC50 RX 851062, 7.25 ± 0.50; pEC50 RX 851062 + 1 µM atipamezole, <5.00. Responses are expressed versus basal [35S]GTPS binding.

Antagonism of (-)-Epinephrine-Mediated [³⁵S]GTPS Binding Response. Antagonism of the (-)-epinephrine (10 nM)-mediated [³⁵S]GTPS binding response by the series of dexefaroxan analogs is illustrated in Fig. 3A at a recombinant G_oCys³⁵¹Ile protein. The capacity of (-)-epinephrine-mediated antagonism was inversely correlated (r² = 0.91, p < 0.001) to the degree of intrinsic activity of the compound (Fig. 3B). Partial agonists were weak antagonists, whereas inverse agonists were the most powerful antagonists of the (-)-epinephrine-mediated [³⁵S]GTPS binding response. (+)-RX 811059, in contrast to RX 851062, decreased the [³⁵S]GTPS binding response to its intrinsic level of negative, inverse agonist activity. The compounds with significant positive efficacy (RX 811046 and, in particular, RX 821008, RX 821010, and RX 821022) were poor blockers of the (-)-epinephrine-mediated [³⁵S]GTPS binding response. The capacity of (-)-epinephrine-mediated antagonism was not related to the gradient of binding affinities at the _2A AR in the copresence of a G_oCys³⁵¹Ile (Fig. 3C). On the other hand, most of the investigated dexefaroxan analogs displayed a comparable antagonist capacity (74 ± 6–91 ± 1%; Fig. 4A) at the G_i2Cys³⁵²Ile protein. A weak relationship (r² = 0.47, p = 0.04) was obtained when the ligand's rank number for antagonism of the (-)-epinephrine-mediated [³⁵S]GTPS binding responses was compared between G_oCys³⁵¹Ile and G_i2Cys³⁵²Ile proteins (Fig. 4B). The capacity of (-)-epinephrine-mediated antagonism was not related to the gradient of binding affinities at the _2A AR in the copresence of a G_i2Cys³⁵²Ile protein (r² = 0.14, p > 0.05; data not shown).

View larger version (26K): [in this window] [in a new window]   Fig. 3. Antagonism of (-)-epinephrine-mediated [35S]GTPS binding response by atipamezole, (+)-RX 811059, dexefaroxan, and its analogs in the copresence of a GoCys351Ile protein. A, CHO-K1 cells were cotransfected with 2A AR and GoCys351Ile protein, treated with PTX (20 ng/ml) and assayed for [35S]GTPS binding responses, as described under Materials and Methods. One micromolar of the indicated ligands was coincubated with 10 nM (-)-epinephrine. Antagonism is expressed as a percentage of the maximal response obtained with 10 nM (-)-epinephrine (84.2 ± 9.3% versus basal [35S]GTPS binding response). Bar graphs are constructed using mean values ± S.E.M. of six independent transfection experiments, each one performed in duplicate. B, relationship between ligands' antagonism of (-)-epinephrine-mediated [35S]GTPS binding response and their intrinsic activity obtained in the absence of (-)-epinephrine. Data were taken from Table 1 and Fig. 3A. A significant correlation (r2 = 0.91, p < 0.001) was obtained. C, lack of relation between ligands' antagonism of (-)-epinephrine-mediated [35S]GTPS binding response and their receptor binding affinity for the 2A AR. Data were taken, respectively, from Table 1 and Fig. 3A.

View larger version (26K): [in this window] [in a new window]   Fig. 4. Antagonism of (-)-epinephrine-mediated [35S]GTPS binding response by atipamezole, (+)-RX 811059, dexefaroxan, and its analogs in the copresence of a Gi2Cys352Ile protein. A, CHO-K1 cells were cotransfected with 2A AR and Gi2Cys352Ile protein and treated as described in the legend to Fig. 3. Antagonism is expressed as a percentage of the maximal response obtained with 10 nM (-)-epinephrine (52.5 ± 0.5% versus basal [35S]GTPS binding response). Bar graphs are constructed using mean values ± S.E.M. of four independent transfection experiments, each one performed in duplicate. Compounds were ranked from the less (RX 821008, rank number 1) to the most active [(+)-RX 811059, rank number 9] antagonist. B, lack of relation between ligands' rank number of antagonism of (-)-epinephrine-mediated [35S]GTPS binding response at GoCys351Ile versus Gi2Cys352Ile protein. Data were taken, respectively, from Figs. 3A and 4A.

Antagonism of UK 14304- and (-)-Epinephrine-Mediated Inhibition of cAMP Formation. The native agonist (-)-epinephrine was not systematically used to monitor inhibition of cAMP formation as mediated by _2A ARs in stably transfected C6-glial cells because it also strongly stimulated cAMP formation (4150 ± 60% versus basal cAMP level) via endogenous ARs. In contrast, UK 14304 (10 nM) inhibited in a PTX-sensitive manner forskolin (100 µM)-stimulated cAMP formation by 65.3 ± 2.3% in the transfected C6-glial cell line (Table 1) as opposed to the naive C6-glial cells. Antagonism of the UK 14304 (10 nM)-mediated inhibition of cAMP formation as performed with the series of dexefaroxan analogs (1 µM) is illustrated in Fig. 5A. One µmolar of (+)-RX 811059 fully antagonized the UK 14304-mediated inhibition of cAMP formation, whereas RX 821010 only weakly (20%) antagonized this response. The rank order of antagonism of UK 14304-mediated inhibition of cAMP formation correlated (r² = 0.89, p < 0.001) with the gradient of binding affinities at the _2A AR (Fig. 5B). Antagonism as displayed by the ligands dexefaroxan and atipamezole was not significantly different (p > 0.05) from that observed with (+)-RX 811059. Because (-)-epinephrine was used to perform [³⁵S]GTPS binding experiments, antagonism of the (-)-epinephrine-mediated inhibition of cAMP formation was further performed in the copresence of 1 µM (+)-propranolol that fully antagonized the stimulation of cAMP formation via endogenous ARs (data not shown). Under this experimental condition, the rank order of antagonism of the (-)-epinephrine-mediated inhibition of cAMP formation fitted (r² = 0.80, p < 0.001) with that of antagonism of the UK 14304-mediated inhibition of cAMP formation (Fig. 5C). In contrast, the antagonist capacity of UK 14304-mediated inhibition of cAMP formation by the herein investigated ligands could not be related to the antagonist capacity of (-)-epinephrine-mediated [³⁵S]GTPS binding responses neither at the G_oCys³⁵¹Ile (Fig. 6) nor at the G_i2Cys³⁵²Ile protein (r² = 0.32, p > 0.05; data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 5. Antagonist effects of atipamezole, (+)-RX 811059, dexefaroxan, and its analogs on UK 14304-mediated inhibition of forskolin-stimulated cAMP formation and the relationship to 2A AR binding affinity. A, stably transfected C6-glial/2A AR cells were coincubated with 100 µM forskolin, 10 nM UK 14304, and putative antagonist at 1 µM for 15 min before cAMP was measured as described under Materials and Methods. Antagonism is expressed as a percentage of the cAMP level obtained with forskolin + UK 14304. Forskolin and forskolin plus UK14304 cAMP levels are 51.11 ± 2.75 and 14.05 ± 1.56 nM, respectively. Bar graphs are constructed using mean values ± S.E.M. of four independent experiments, each one performed in triplicate. B, relationship between ligands' antagonism of UK 14304-mediated inhibition of forskolin-stimulated cAMP formation and their [3H]RX 821002 binding affinity for the 2A AR. [3H]RX 821002 binding data were obtained in transfected CHO-K1 cells, as described under Materials and Methods. Mean values ± S.E.M. from three independent experiments are summarized in Table 1. A significant correlation (r2 = 0.89, p < 0.001) was obtained. C, relationship between ligands' antagonism of UK 14304-mediated inhibition of forskolin-stimulated cAMP formation and ligands' antagonism of (-)-epinephrine-mediated inhibition of forskolin-stimulated cAMP formation. A significant correlation (r2 = 0.80, p < 0.001) was obtained. Data for UK 14304-mediated cAMP formation were taken from Fig. 4A. Data for (-)-epinephrine-mediated cAMP formation were taken from two experiments made in the presence of 1 µM propranolol to inhibit the endogenous AR-mediated cAMP response.

View larger version (29K): [in this window] [in a new window]   Fig. 6. Lack of relation between ligands' antagonism of (-)-epinephrine-mediated [35S]GTPS binding response in the copresence of a GoCys351Ile protein and ligands' antagonism of UK 14304-mediated inhibition of forskolin-stimulated cAMP formation. Data were taken respectively from Figs. 3A and 5A.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
The present report investigated the pharmacological responses for a series of putative adrenergic antagonists chemically analogous to the imidazoline derivative dexefaroxan at two different _2A AR states: 1) an enhanced high-affinity _2A AR* state upon coexpression with a recombinant G_oCys³⁵¹Ile protein and by monitoring [³⁵S]GTPS binding responses at CHO-K1 cellular membranes in the absence of sodium ions, and 2) a presumably uncoupled _2A AR state as monitored either upon coexpression with a recombinant G_i2Cys³⁵²Ile protein and by monitoring [³⁵S]GTPS binding responses as described above, or in C6-glial cells at the level of adenylate cyclase inhibition mediated by endogenous G_i/o proteins. In contrast to dexefaroxan, its analogs were devoid of intrinsic activity at the wild-type _2A AR when monitoring their cAMP response. Otherwise, several of these ligands demonstrated at nanomolar concentrations either positive or negative efficacy in the presence of a recombinant PTX-resistant G_oCys³⁵¹Ile protein, whereas they behaved as weak partial agonist or silent compounds at a G_i2Cys³⁵²Ile protein. This confirms previous data indicating that a mutant Thr³⁴³Lys _2A AR in the copresence of a G_i2Cys³⁵²Ile protein did not generate constitutive activation (Pauwels et al., 2000). The degree of intrinsic activity for a given ligand mainly depends on the G protein affinity state in which the receptor is thermodynamically stabilized (Kenakin, 2001). By coexpressing the _2A AR with a mutant G_oCys³⁵¹Ile protein, but not with a G_i2Cys³⁵²Ile protein, an enhanced high-affinity _2A AR* state was achieved. Hence, receptor constitutive activation was increased; this could be attenuated by some of the dexefaroxan analogs acting as inverse agonists at the _2A AR. The [³⁵S]GTPS binding assay was performed in the absence of sodium ions, an experimental condition that has been predicted mathematically (Costa et al., 1992) to enhance the relative efficacies of both agonists and inverse agonists. Under this assay condition, both negative and positive efficacies were magnified compared with the recordings at the cAMP level. On the other hand, the mutant G_i2Cys³⁵²Ile protein was coexpressed with a wt _2A AR to evaluate the antagonist activity of dexefaroxan analogs under experimental conditions where no precoupled _2A AR states could be observed. The herein [³⁵S]GTPS binding assays are, therefore, useful to monitor _2A AR responses under both high- and low-affinity _2A AR states dependent on the coexpressed recombinant G protein.

The dexefaroxan analogs RX 811059 and RX 851062 displayed the most pronounced inverse agonist responses in the herein investigated series. RX 831003 has been reported to act as a protean agonist (Pauwels et al., 2002), yielding partial agonism at the mutant Thr³⁷³Lys _2A AR in the copresence of a G₁₅ protein and partial inverse agonism in the copresence of a G_oCys³⁵¹Ile protein. RX 851057 and the distantly related imidazole derivative atipamezole behaved as neutral, silent antagonists. The intrinsic activity of atipamezole seems dependent on the assay and/or readout systems. It displayed inverse agonist activity at endogenous _2A AR in human erythroleukemia cells (HEL 92.1.7) by following both Ca²⁺ elevation and cAMP production (Jansson et al., 1998), whereas it remained a silent antagonist at an _2A AR carrying an activating Thr³⁷³Lys mutation (Pauwels et al., 2000). A dichloro-substituted imidazoline derivative RX 821010 displayed high-efficacy agonist properties almost similar to (-)-epinephrine. The reversal of both negative and positive responses by atipamezole and at a potency relevant to its binding affinity further confirms that the observed ligand responses were mediated by the _2A AR. The rank order of the ligands' capacity to antagonize (-)-epinephrine-mediated [³⁵S]GTPS binding response was inversely correlated to the degree of intrinsic activity of the compounds and without relation to their binding affinity for the _2A AR. Efficacious agonists such as (-)-epinephrine have been proposed (Kenakin, 1995) to generate multiple activated _2A AR conformations being equally susceptible to activate both G_i and G_s proteins. On the other hand, partial agonists (i.e., oxymetazoline) may produce a unique activated receptor form that couples only to G_i proteins (Kenakin, 1995). The herein investigated [³⁵S]GTPS binding responses are likely to be exclusively mediated by the recombinant G_oCys³⁵¹Ile protein because signaling via endogenous G proteins was abolished by PTX. In addition, blockade of (-)-epinephrine-mediated [³⁵S]GTPS binding responses were monitored in the copresence of agonist and putative antagonist at the _2A AR. The resulting _2A AR conformation was therefore codetermined by both (-)-epinephrine and by the partial agonist or partial inverse agonist to activate one single G_oCys³⁵¹Ile protein subtype. The _2A AR subtype has been shown to activate both G_i/o and G_s proteins resulting in opposing effects at the adenylate cyclase enzyme (Eason et al., 1992, 1994). Although both (-)-epinephrine and UK 14304 stimulated adenylate cyclase efficaciously, their potency to activate this pathway was in the micromolar range (Eason et al., 1992). Thus, it is unlikely that stimulation of G_s occurred in the herein described cAMP assay in which 10 nM of each agonist was used, thereby representing the inhibitory action of G_i protein activation.

The degree of intrinsic activity for a given ligand, especially for partial agonists, also depends on whether the output response is proximal to the receptor signaling cascade (i.e., [³⁵S]GTPS binding) or distal to it (i.e., adenylate cyclase activity) (Kenakin, 2001). The herein observed rank order for the capacity of antagonism of the UK 14304-mediated inhibition of cAMP formation highly correlated with ligands' binding affinities for the _2A AR, strongly suggesting that antagonism is mediated via the _2A AR. On the other hand, no relationship was observed between the antagonist capacities for the series of dexefaroxan analogs for both agonist-mediated [³⁵S]GTPS and cAMP responses. These data suggest differential signaling and/or blockade for these compounds that are assumed to be antagonists at the _2A AR. A difference in the antagonist capacity toward cAMP and [³⁵S]GTPS binding responses may reflect that antagonists can preferentially block selective signaling pathways. The investigated putative antagonists, differing in their chemical structure, are likely to stabilize different _2A AR conformations in case they yield intrinsic activity, as it was already reported for agonists, such as clonidine and oxymetazoline (Salminen et al., 1999). (-)-Epinephrine, UK 14304, oxymetazoline, and clonidine have been demonstrated to act as full agonists with respect to the G_o1 protein (Yang and Lanier, 1999), but oxymetazoline and clonidine were partial agonists with respect to G_i1 protein activation. The results in the present study extend this notion for ligands that are assumed to be antagonists at one single effector pathway. C6-glial cells predominantly express endogenous G_i2 over G_i3 proteins, whereas no G_o protein immunoreactivity was detectable (Charpentier et al., 1993). Given the herein presented absence of relationship on antagonism of the (-)-epinephrine-mediated [³⁵S]GTPS binding response in the copresence of a G_i2Cys³⁵²Ile protein and cAMP response, we can assume that G_i3 protein may participate to the _2A AR-mediated cAMP response in C6-glial cells.

In conclusion, the herein presented results support the notion of differential pharmacological responses through a single _2A AR subtype for putative adrenergic antagonists. Agonist-stimulated _2A ARs can adopt different conformations in response to such an "antagonist", which may translate into distinct pharmacological responses mainly dependent on the investigated receptor-activated effector pathway. This study also suggests that neutral antagonists are probably rare. Pharmacological diversity via a single receptor subtype may thereby be relevant and also add to the therapeutic value of this class of _2A AR molecules.

	Acknowledgements

 
We sincerely thank Claudie Cathala and Fabrice Lestienne for the construction of plasmids and Stéphanie Brignatz for secretarial assistance.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.102.048215.

ABBREVIATIONS: AR, adrenoceptor; wt, wild-type; dexefaroxan, R-(+)-2-(2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; GTPS, guanosine 5'-O-thiotriphosphate; PTX, Bordetella pertussis toxin; CHO, Chinese hamster ovary; RX 821002 1,4-[6,7(n)-benzodioxan-2-methoxy-2-yl)-4,5-dihydro-1H-imidazole; UK 14304, 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline tartrate; RX 811059, 2-(2-ethoxy-2,3-dihydro-benzo[1,4]dioxin-2-yl)-4,5-dihydro-1H-imidazole; RX 821008, 2-(4-chloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 831001, 2-(2-n-propyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 831003, 2-(2-n-pentyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 851057, 2-(4-hydroxy-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 851062, 2-(4-methoxy-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 841047, 2-(5-chloro-2-ethyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 821010, 2-(6,7-dichloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 811046, 2-(6-methyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 811022, 2-(5-methyl-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 811012, 2-(5-chloro-2,3-dihydrobenzofuran-2-yl)-4,5-dihydro-1H-imidazole; RX 811055, 2-(5,6-dimethyl-2,3-dihydrobenzofuran-2-yl)-4,5-duhydro-1H-imidazole.

Address correspondence to: Dr. Peter Pauwels, Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17, avenue Jean Moulin, 81106 Castres Cédex, France. E-mail. peter.pauwels{at}pierre-fabre.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Airriess CN, Rudling JE, Midgley JM, and Evans PD (1997) Selective inhibition of adenylyl cyclase by octopamine via a human cloned 2A-adrenoceptor. Br J Pharmacol 122: 191–198.[CrossRef][Medline]

Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.[CrossRef][Medline]

Bylund DB, Eikenberg DC, Hieble JP, Langer SZ, Lefkowitz RJ, Minneman KP, Molinoff PB, Ruffolo RR Jr, and Trendelenburg U (1994) IV International Union of Pharmacology nomenclature of adrenoceptors. Pharmacol Rev 46: 121–136.[Medline]

Chapleo CB, Myers PL, Butler RC, Davis JA, Doxey JC, Higgins SD, Myers M, Roach AG, Smith CF, Stillings MR, et al. (1984) -Adrenoreceptor reagents. 2. Effects of modification of the 1,4-benzodioxan ring system on -adrenoreceptor activity. J Med Chem 27: 570–576.[CrossRef][Medline]

Chapleo CB, Myers PL, Butler RC, Doxey JC, Roach AG, and Smith CF (1983) Alpha-adrenoreceptor reagents. 1. Synthesis of some 1,4-benzodioxans as selective presynaptic 2-adrenoreceptor antagonists and potential antidepressants. J Med Chem 26: 823–831.[CrossRef][Medline]

Charpentier N, Prézeau L, Carrette J, Bertorelli R, Le Cam G, Manzoni O, Bockaert J, and Homburger V (1993) Transfected G_o1 inhibits the calcium dependence of -adrenergic stimulated cAMP accumulation in C6-glioma cells. J Biol Chem 268: 8980–8989.[Abstract/Free Full Text]

Costa T, Ogino Y, Munson P, Onaran H, and Rodbard D (1992) Drug efficacy at guanine nucleotide-binding regulatory protein-linked receptors: thermodynamic interpretation of negative antagonism and of receptor activity in the absence of ligand. Mol Pharmacol 41: 549–560.[Abstract]

Dupuis DS, Tardif S, Wurch T, Colpaert FC, and Pauwels PJ (1999) Modulation of 5-HT_1A receptor signaling by point-mutation of cysteine³⁵¹ in the rat G_o protein. Neuropharmacology 38: 1035–1041.[CrossRef][Medline]

Eason MG, Jacinto MT, and Liggett SB (1994) Contribution of ligand structure to activation of 2-adrenergic receptor subtype coupling to Gs. Mol Pharmacol 45: 696–702.[Abstract]

Eason MG, Kurose H, Holt BD, Raymond JR, and Liggett SB (1992) Simultaneous coupling of 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of 2C10, 2C4 and 2C2 adrenergic receptors to G_i and G_s. J Biol Chem 267: 15795–15801.[Abstract/Free Full Text]

Eason MG and Liggett SB (1993) Human 2-adrenergic receptor subtype distribution: widespread and subtype-selective expression of 2C10, 2C4 and 2C2 mRNA in multiple tissues. Mol Pharmacol 44: 70–75.[Abstract]

Handy DE, Flordellis CS, Bogdanova NN, Bresnahan MR, and Gavras H (1993) Diverse tissue expression of rat alpha 2-adrenergic receptor genes. Hypertension 21: 861–865.[Abstract/Free Full Text]

Jansson CC, Kuhkonen JP, Nasman J, Huifang G, Wurster S, Virtanen R, Savola JM, Cockcroft V, and Akermen KE (1998) Protean agonism at 2A-adrenoceptors. Mol Pharmacol 53: 963–968.[Abstract/Free Full Text]

Kenakin T (1995) Agonist-receptor efficacy. II. Agonist trafficking of receptor signals. Trends Pharmacol Sci 16: 232–238.[CrossRef][Medline]

Kenakin T (2001) Inverse, protean and ligand-selective agonism: matters of receptor conformation. FASEB J 15: 598–611.[Abstract/Free Full Text]

Kukkonen JP, Jansson CC, and Åkerman KE (2001) Agonist trafficking of G(i/o)-mediated (2A)-adrenoceptor responses in HEL 92.1.7 cells. Br J Pharmacol 132: 1477–1484.[CrossRef][Medline]

Lakhlani PP, Lovinger DM, and Limbird LE (1996) Genetic evidence for involvement of multiple effector systems in 2A-adrenergic receptor inhibition of stimulus-secretion coupling. Mol Pharmacol 50: 96–103.[Abstract]

Limbird LE (1988) Receptors linked to inhibition of adenylate cyclase: additional signaling mechanisms. FASEB J 2: 2686–2695.[Abstract]

Marjamäki A, Frang H, Pihlavisto M, Hoffren AM, Salminen T, Johnson MS, Kallio J, Javitch JA, and Scheinin M (1999) Chloroethylclonidine and 2-aminoethyl methanethiosulfonate recognize two different conformations of the human (2A)-adrenergic receptor. J Biol Chem 274: 21867–21872.[Abstract/Free Full Text]

Martel JC, Chopin P, Colpaert FC, and Marien M (1998) Neuroprotective effect of the ₂-adrenoceptor antagonists, (+)-efaroxan and (±)-idazoxan, against quinolinic acid-induced lesions in rat striatum. Exp Neurol 154: 595–601.[CrossRef][Medline]

Pauwels PJ and Colpaert FC (2000) Disparate ligand-mediated Ca²⁺ responses by wt, mutant Ser²⁰⁰Ala and Ser²⁰⁴Ala _2A-adrenoceptor: G₁₅ fusion proteins: evidence for multiple ligand-activation binding sites. Br J Pharmacol 130: 1505–1512.[CrossRef][Medline]

Pauwels PJ, Rauly I, Wurch T, and Colpaert FC (2002) Evidence for protean agonism of RX831003 at 2A-adrenoceptors by co-expression with different G protein subunits. Neuropharmacology 42: 855–863.[CrossRef][Medline]

Pauwels PJ, Tardif S, Wurch T, and Colpaert FC (2000) Facilitation of constitutive _2A-adrenoceptor activity by both single amino acid mutation Thr³⁷³Lys and G_o protein coexpression: evidence for inverse agonism. J Pharmacol Exp Ther 292: 654–663.[Abstract/Free Full Text]

Pauwels PJ, Wurch T, Palmier C, and Colpaert FC (1996) Pharmacology of cloned human 5-HT_1D receptor-mediated functional responses in stably transfected rat C6-glial cell lines: further evidence differentiating human 5-HT_1D and 5-HT_1B receptors. Naunyn-Schmiedeberg's Arch Pharmacol 353: 144–156.[Medline]

Pauwels PJ, Wurch T, Tardif S, Finana F, and Colpaert FC (2001) Analysis of ligand activation by _2C-adrenoceptor as compared to _2A and _2B-adrenoceptors at a similar receptor:G protein density ratio using fusion proteins. Naunyn-Schmiedeberg's Arch Pharmacol 363: 526–536.[CrossRef][Medline]

Rudling JE, Kennedy K, and Evans PD (1999) The effect of site-directed mutagenesis of two transmembrane serine residues on agonist-specific coupling of a cloned human 2A-adrenoceptor to adenylyl cyclase. Br J Pharmacol 127: 877–886.[CrossRef][Medline]

Salminen T, Varis M, Nyronen T, Pihlavisto M, Hoffren AM, Lonnberg T, Marjamaki A, Frang H, Savola JM, Scheinin M, et al. (1999) Three-dimensional models of (2A)-adrenergic receptor complexes provide a structural explanation for ligand binding. J Biol Chem 274: 23405–23413.[Abstract/Free Full Text]

Small KM and Liggett SB (2001) Identification and functional characterization of (2)-adrenoceptor polymorphisms. Trends Pharmacol Sci 22: 471–477.[CrossRef][Medline]

Tavares A, Handy DE, Bogdanova NN, Rosene DL, and Gavras H (1996) Localization of 2A- and 2B-adrenergic receptor subtypes in brain. Hypertension 27: 449–455.[Abstract/Free Full Text]

Tellez S, Colpaert FC, and Marien M (1999) ₂-Adrenoceptor modulation of cortical acetylcholine release in vivo. Neuroscience 84: 1041–1050.

Yang Q and Lanier SM (1999) Influence of G protein type on agonist efficacy. Mol Pharmacol 56: 651–656.[Abstract/Free Full Text]

Wade SM, Lan K-L, Moore DJ, and Neubig RR (2001) Inverse agonist activity at the _2A-adrenergic receptor. Mol Pharmacol 59: 532–542.[Abstract/Free Full Text]

Wang CD, Buck MA, and Fraser CM (1991) Site-directed mutagenesis of alpha 2A-adrenergic receptors: identification of amino acids involved in ligand binding and receptor activation by agonists. Mol Pharmacol 40: 168–179.[Abstract]

Wurch T, Colpaert FC, and Pauwels PJ (1999) G-protein activation by putative antagonists at mutant Thr³⁷³Lys _2A adrenergic receptors. Br J Pharmacol 126: 939–948.[CrossRef][Medline]

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